This invention relates to nucleoside crosslinking agents and to the use of these compounds in the preparation of oligonucleotides. It also relates to derivatives of pyrazolo [3,4-d] pyrimidine which are useful as nucleic acid bases for the preparation of oligonucleotides.
Oligonucleotides are useful as diagnostic probes for the detection of xe2x80x9ctargetxe2x80x9d DNA or RNA sequences. In the past, such probes were made up of sequences of nucleic acid containing purine, pyrimidine or 7-deazapurine nucleotide bases (U.S. Pat No. 4,711,955; Robin et al., J. Can. J. Chem, 60:554 (1982); Robins et al., J. Org. Chem., 48:1854 (1983)). The method for attaching chemical moieties to these bases has been via an acetoxy-mercuration reaction. which introduce, covalently bound mercury atoms into the 5-position of the pyrimidine ring, the C-8 position of the purine ring or the C-7 position of a 7-deazapurine ring (Dale et al., Proc. Natl. Acad. Sci. USA, 70:2238 (1973); Dale et al., Biochemistry, 14:2447 (1975)), or by the reaction of organomercurial compounds with olefinic compounds in the presence of palladium catalysts (Ruth et al., J. Org. Chem., 43:2870 (1978); Bergstrom et al., J. Am. Chem. Soc., 100:8106 (1978); Bigge et al., J. Am. Chem. Soc., 102:2033 (1980)).
The sugar component of oligonucleotide probes has been, until the present, composed of nucleic acid containing ribose or deoxyribose or, in one case, natural xcex2-arabinose (patent publication EP 227,459).
A novel class of nucleotide base, the 3,4-disubstituted and 3,4,6-trisubstituted pyrazolo[3,4-d]-pyrimidines, has now been found which offers several advantages over the prior art. The de novo chemical synthesis of the pyrazolopyrimidine and the resulting nucleotide allows for the incorporation of a wide range of functional groups in a variety of different positions on the nucleotide base and for the use of different sugar moieties. Also, adenine, guanine and hypoxanthine analogs we obtained from a single nucleoside precursor. Additionally, the synthesis does not require the use of toxic heavy metals or expensive catalysts. Similar pyrazolo [3,4-d] pyrimidines are known (Kobayashi. Chem. Pharm. Bull., 21:941 (1973)); however, the substituents on the group are different from those of the present invention and their only use is as xanthine oxidase inhibitors. The concept of crosslinkable nucleoside probes for use in therapeutic and diagnostic applications is related to the pioneering work of B. R. Baker, xe2x80x9cDesign of Active-Site-Directed Irreversible Enzyme inhibitors.xe2x80x9d Wiley, N.Y., (1967), who used who was termed xe2x80x9cactive-site-directed enzyme inhibitorsxe2x80x9d in chemotherapeutic applications.
In recent years, the concept of incorporating a crosslink in an oligonucleotide has been sporadically discussed in efforts to develop superior sequence probes. Knorre and Vlassov, Prog. Nucl. Acid Res. Mol. Biol., 32:291 (1985), have discussed sequence-directed cross-linking (xe2x80x9ccomplementary addressed modificationxe2x80x9d) using an N-(2-chloroethyl)-N-methylaniline group attached to either the 3xe2x80x2- or 5xe2x80x2-terminus of oligonucleotides. Summerton and Bartlett, J. Mol. Biol., 122:145 (1978) have shown that an 8-atom chain, attached to a cytosine residue at its C-4 position and laminating in the highly reactive bromomethyl ketone group, can crosslink to the N-7 of guanosine.
Webb and Matteucci, Nucleic Acids Res., 14:7661(1986). have prepared oligonucleotides containing a 5-methyl-N,N-ethanocytosine base which is capable of slow cross linking with a complementary strand. In a conceptually related alkylation via a linker arm within a DNA hybrid, Iverson and Dervan, Proc. Natl. Acad, Sci. USA, 85:4615 (1988), have shown opposite strand methylation, triggered by BrCN activation of a methylthio ether, predominately on a guanine base located two pairs from the base bearing the linker.
Oligonucleotides may be used as chemotherapeutic agents to control the expression of gene sequences unique to an invading organism, such as a virus, a fungus, a parasite or a bacterium. In nature, some RNA expression in bacteria is controlled by xe2x80x9cantionsexe2x80x9d RNA, which exerts its effect by forming RNA:RNA hybrids with complementary target RNAs and modulating or inactivating their biological activity. A variety of recent studies using plasmid vectors for the introduction of antisense RNAs into eukaryotic cells have shown land they effectively inhibit expression of MRNA targets in vivo (reviewed in Green, et at., Ann. Rev. Biochem., 55: 569-597 (1986)). Additionally, a specific mRNA amongst a large number of mRNAs can be selectively inactivated for protein synthesis by hybridization with a complementary DNA restriction fragment which binds to the mRNA and prevents its translation into protein on ribosomes (Paterson, et al., Proc. Natl. Acad. Sci. 74; 4370-4374 (1977); Hastie et al., Proc. Natl. Acad. Sci., 75: 1217-1221 (1978)).
In the first demonstration of the concept of using sequence-specific, antisense oligonucleotides as regulators of gene expression and as chemotherapeutic agents. Zameonik and Stephenson, Proc. Natl. Acad. Sci. USA, 75:280 (1978), showed the a small antisense oligodeoxynucleotide probe can inhibit replication of Rous Sarcoma virus in cell culture and that RSV viral RNA translation is inhibited under these conditions (Stephenson et al., Pro. Natl. Acad. Sci. USA 75:285 (1978)). Zamecnik et al., Proc. Natl. Acad. Sci. USA, 83:4143 (1986), have also shown that oligonucleotides complementary to portions of the HIV genome are capable of inhibiting protein expression and virus replication in cell culture. Inhibition of up to 95% was obtained with oligonucleotide concentration of about 70 xcexcM. Importantly, they showed with labeled phosphate studies that the oligonucleotides enter cells intend and are reasonably stable to metabolism.
Uncharged methylphosphonate oligodeoxynucleotides with a sequence complementary to the initiation colon regions of rabbit globin mRNA inhibited the translation of the mRNA in both cell-free systems and in rabbit reticulocytes (Blake et al., Biochemistry 24:6139 (1985)). Another unchanged methylophosphonate oligonucleotide analog, an 8-nucleotide sequence complementary to the acceptor splice junction of a mRNA of Herpes simply virus, Type 1, can inhibit virus replication in intact Vero cells. However, fairly high concentrations ( greater than 25 mM) of this nonionic probe were required for this inhibition.
Although the impact of crosslinking oligonucleotides in the chemotherapeutic field might be of great significance, their impact in DNA probe-based diagnostics is of equally great importance. The ability in covalently crosslink probe-target hybrids has the potential to dramatically improve background and sensitivity limits in diagnostic assays as well as permit novel assay funnels. Specific innovations (discussed previously by Gamper et al., Nucl. Acids Res., 14, 9943 (1988)) include:
(a) incorporation of a denaturing wash step to remove background;
(b) use of the crosslink as an additional tier of discrimination;
(c) crosslinking occurring at or near the melting temperature of the expected hybrid to insure exquisite specificity and to substantially reduce secondary structure in the target, thereby increasing the efficiency of hybrid formation; and
(d) novel solution hybridization formats as exemplified by the Reverse Southern protocol.
The concept of crosslinking, however, suggests potential problems that must be circumvented. For instance, the oligonucleotide containing a crosslinking arm might covalently bond to the target sequence so readily that mismatching of sequences will occur, possibly resulting in host toxicity. On the other hand, the crosslinking reaction must be fast enough to occur before correctly matched sequences can dissociate.
This issue can be addressed by constructing an oligonucleotide that, upon hybridization, results in a duplex whose Tm is just above the physiological temperature of 37xc2x0 C. Thus, even a single mismatched base will prevent hybrid formation and therefore crosslinkage. The optimization can be accomplished by judicious choice of oligonucleotide length and base composition, as well as position of the modified base within the probe. The probe must be long enough, however, to insure specific targeting of a unique site.
European Patent Application No. 86309090.8 describes the formation of chemically modified DNA probes such as 5-substituted uridinyl in which the substituent does not crosslink but contains a chemical or physical reporter group. WO8707611 describes a process for labeling DNA fragments such as by chemically modifying the fragment followed by reaction with a fluorescent dye. Yabusaki et al., in U.S. Pat. No. 4,599,303 disclose a scheme for covalently crosslinking oligonucleotides such as by formation of furocoumarin monoadducts of thymidine which are made to covalently bond to other nucleotides upon photoexcitation. EP 0259186 describes adducts of macromolecules and biotin which can be used as crosslinking nucleic acid hybridization probes. WO9503075 describes crosslinking disulfonic eaters useful as nucleic acid fragmentation agents. DE3310337 describes the covalent crosslinking of single-stranded polynucleotides to such macromolecules as proteins with the resulting complex subsequently used as a marker in hybridization experiments in the search for complementary sequences in foreign polynucleotides
A need exists for probe oligonucleotides, consisting of sufficient base sequences to identify target sequences with high specificity, that are provided with one or more crosslinking arms which readily form covalent bonds with specific complementary bases. Such oligonucleotides may be used as highly selective probes in hybridization assays. The oligonucleotides may also be used as antisensing agents of RNAs, e.g., to chemotherapy.
This invention is directed to crosslinking agents which accomplish crosslinking between specific sites on adjoining strands of oligonucleotides. The crosslinking reaction observed is of excellent specificity. The invention is also directed to oligonucleotides comprising at least one of these crosslinking agents and to the use of the resulting novel oligonucleotides for diagnostic and therapeutic purposes.
More particularly, the crosslinking agents of this invention are derivatives a nucleotide bases with a crosslinking arm and are of the following formula (xcex93):
R1xe2x80x94Bxe2x80x94(CH2)qxe2x80x94(Y)xe2x80x94(CH2)mxe2x80x94Axe2x80x2xe2x80x83xe2x80x83(xcex93)
wherein,
R1 is hydrogen, or a sugar moiety a analog thereof optionally substituted at its 3xe2x80x2 or its 5xe2x80x2 position with a phosphorus derivative attached to the sugar moiety by an oxygen end including groups Q1 Q2 and Q3, or with a reactive precursor thereof suitable for nucleotide bond formation;
Q1 is hydroxy, phosphate or diphosphate;
Q2 isxe2x95x90of or xe2x95x90S;
Q3 is CH2xe2x80x94Rxe2x80x2, Sxe2x80x94Rxe2x80x2, Oxe2x80x94Rxe2x80x2, or Nxe2x80x94Rxe2x80x2Rxe2x80x3;
each of Rxe2x80x2 and Rxe2x80x3 is independently hydrogen or C1-6 alkyl;
B is a nucleic acid base or analog thereof that is a component of an oligonucleotide;
Y is a functional linking group;
each of m and q is independently 0 to 8, inclusive;
r is 0 or 1; and
Axe2x80x2 is a leaving group.
The invention also provides novel oligonucleotides comprising to least one of the above nucleotide base derivatives of formula xcex93.
Nucleotides of this invention and oligonucleotides into which the nucleotides have been incorporated may be used as probes. Since probe hybridization is reversible, albeit slow, it is desirable to ensure that each time a probe hybridize, with the current target sequence, the probe is irreversibly attached to that sequence. The covalent crosslinking arm of the nucleotide bases of the present invention will prominently modify the target strand, or cause depurination. As such, the oligonucleotides of this invention are useful in the identification, isolation, localization and/or detection of complementary nucleic acid sequences of interest in cell-free and cellular systems. Therefore, the invention further provides a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucleotide probe comprising at least one of a labeled nucleotide base of the present invention.
This invention also provides novel substituted pyrazolo [3,4-d] pyrimidines which are useful as a nucleotide base in preparing nucleosides and nucleotides, rather than the natural purine or pyrimidine bases or the deazapurine analogs.